Multi-stage acoustophoresis device

ABSTRACT

An acoustophoresis device made up of modular components is disclosed. Several modules are disclosed herein, including ultrasonic transducer modules, input/output modules, collection well modules, and various connector modules. These permit different systems to be constructed that have appropriate fluid dynamics for separation of particles, such as biological cells, from a fluid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 14/533,753, filed on Nov. 5, 2014. U.S. patent application Ser.No. 14/533,753 claimed priority to U.S. Provisional Patent ApplicationSer. No. 61/900,635, filed Nov. 5, 2013, and to U.S. Provisional PatentApplication Ser. No. 62/020,088, filed on Jul. 2, 2014. This applicationalso claims priority to U.S. Provisional Patent Application Ser. No.62/322,262, filed on Apr. 14, 2016, and to U.S. Provisional PatentApplication Ser. No. 62/307,489, filed on Mar. 12, 2016, and to U.S.Provisional Patent Application Ser. No. 62/235,614, filed on Oct. 1,2015. The disclosures of these applications are hereby fullyincorporated by reference in their entirety.

BACKGROUND

The ability to separate a particle/fluid mixture into its separatecomponents is desirable in many applications. Physical size exclusionfilters can be used for this purpose, where the particles are trapped onthe filter and the fluid flows through the filter. Examples of physicalfilters include those that operate by tangential flow filtration, depthflow filtration, hollow fiber filtration, and centrifugation. However,physical filters can be complicated to work with. For instance, as thephysical filter fills up, filtration capacity is reduced. Also, usingsuch filters requires periodic stopping to remove the filter and obtainor clear the particles trapped thereon.

Acoustophoresis is the separation of particles using high intensitysound waves, and without the use of membranes or physical size exclusionfilters. It has been known that high intensity standing waves of soundcan exert forces on particles. A standing wave has a pressure profilewhich appears to “stand” still in time. The pressure profile in astanding wave contains areas of net zero pressure at its nodes andanti-nodes. Depending on the density and compressibility of theparticles, they will be trapped at the nodes or anti-nodes of thestanding wave. However, conventional acoustophoresis devices have hadlimited efficacy due to several factors including heat generation,limits on fluid flow, and the inability to capture different types ofmaterials. Improved acoustophoresis devices using improved fluiddynamics would be desirable.

BRIEF SUMMARY

The present disclosure relates to modular components that can be used tobuild acoustophoretic systems with improved fluid dynamics that can beused to improve separation of particles from a particle/fluid mixture.Either a new mixture with an increased concentration of particles isobtained, or the particles themselves can be obtained or a clarifiedfluid containing biomolecules, such as recombinant proteins ormonoclonal antibodie, may be produces. In more specific embodiments, theparticles are biological cells, such as Chinese hamster ovary (CHO)cells, NSO hybridoma cells, baby hamster kidney (BHK) cells, or humancells; lymphocytes such as T cells (e.g., regulatory T-cells (Tregs),Jurkat T-cells), B cells, or NK cells; their precursors, such asperipheral blood mononuclear cells (PBMCs); algae or other plant cells,bacteria, viruses, or microcarriers. Several different types of modulesand overall systems are described herein.

Disclosed in various embodiments herein are modular acoustophoresisdevices, comprising an ultrasonic transducer module. The ultrasonictransducer module comprises: a housing defining a primary flow channelbetween a first end and a second end of the housing; at least oneultrasonic transducer located on a side of the housing; at least onereflector located on the side of the housing opposite the at least oneultrasonic transducer; an first attachment member at the first end ofthe housing; and a second attachment member at the second end of thehousing which may be complementary to the first attachment member.

The first attachment member and the second attachment member of theultrasonic transducer module may operate by press-fitting or screwing.The attachment members are used to cooperatively fix or fit together thevarious modules and construct the overall acoustophoretic device.

Some embodiments of the ultrasonic transducer module further include aport on a side of the housing between the transducer and the reflector.

Also disclosed are collection well modules comprising: a housing havinga well that tapers downwards in cross-sectional area from a single inletto a vertex, and a drain line connecting the vertex to a port on a sideof the housing; and an attachment member at the inlet, the attachmentmember adapted to connect the collection well module to the ultrasonictransducer module.

In particular embodiments, the attachment member of the collection wellmodule is complementary to the second attachment member of theultrasonic transducer module.

Also disclosed are angled collection well modules comprising: a housinghaving a first opening and a second opening that lead into a common wellthat taper downwards in cross-sectional area to a vertex, and a drainline connecting the vertex to a port on a side of the housing; a firstattachment member at the first opening adapted to connect the collectionwell module to the ultrasonic transducer module; and a second attachmentmember at the second opening adapted to connect the collection wellmodule to the ultrasonic transducer module; wherein the first opening islocated at an acute angle relative to a base of the housing.

The second opening may be located on the housing opposite the base ofthe housing. The first attachment member can be complementary to thesecond attachment member.

Alternative embodiments of a U-turn inlet/outlet module are alsodisclosed herein, comprising: a housing having an upper end and a lowerend; a flow channel having a first end and a second end; an inlet portand an outlet port at the first end of the flow channel; an openingdefining the second end of the flow channel and located at the lower endof the housing; and an attachment member at the lower end of thehousing, the attachment member adapted to connect the inlet/outletmodule to the ultrasonic transducer module; wherein the flow channel isshaped such that fluid flows from the inlet port through the opening andthen to the outlet port.

In particular embodiments, the inlet port and the outlet port are spacedfrom each other on a common side of the housing.

The U-turn inlet/outlet module can further comprise a wall located inthe flow channel between the inlet port and the outlet port. The wallcan be placed so that a cross-sectional area of the flow channel for theinlet port is smaller than a cross-sectional area of the flow channelfor the outlet port. Sometimes, the wall extends out of the opening atthe lower end of the housing. In other embodiments, the wall is spacedapart from the upper end of the housing so as to form a pressure reliefpassage between the inlet port and the outlet port.

In some embodiments, the inlet port and the outlet port are spaced apartfrom the upper end of the housing such that fluid must flow from theinlet port towards the upper end over a primary retainer wall beforeexiting through the opening at the lower end of the housing.

In other embodiments, the inlet port and the second port are located atthe upper end of the housing, and the flow channel is in the shape oftwo tubes, one tube leading to the inlet port and the other tube leadingto the outlet port.

Also disclosed herein are port modules comprising: a housing defining asingle flow channel between an upper end and a lower end of the housing;and an attachment member at the lower end of the housing, the attachmentmember adapted to connect the port module to the ultrasonic transducermodule.

Certain connector modules are also disclosed which comprise: a housinghaving an upper end, a lower end, and a side; a first opening on theupper end of the housing; a second opening on the side of the housing, aflow channel being defined between the first opening and the secondopening; a first attachment member at the upper end of the housing; anda second attachment member at the side of the housing which may becomplementary to the first attachment member.

In some particular embodiments, the connector module further comprises:a third opening on the lower end of the housing, the flow channel alsojoining the first opening and the second opening to the third opening;and a third attachment member at the lower end of the housing which iscomplementary to the first attachment member.

Also disclosed are other connector modules comprising: a housing havingan upper end, a lower end, and a side; a first opening on the upper endof the housing; a second opening on the lower end of the housing, astraight flow channel being defined between the first opening and thesecond opening; a first attachment member at the upper end of thehousing; and a second attachment member at the lower end of the housing,wherein the first attachment member is the same as the second attachmentmember. These particular connector modules are intended to permit theorientation of a given opening on a different module to be reversed, ifneeded.

In some embodiments, the first attachment member and the secondattachment member are both female members. In other embodiments, thefirst attachment member and the second attachment member are both malemembers.

Also disclosed is a variable-volume collection well module comprising: ahousing having a well with a constant cross-section, an inlet at anupper end, and a bottom end; and a plunger that provides a floor to thewell, the plunger adapted to move through the well from the bottom endtowards the upper end. In some further embodiments, this module furtherincludes a port that is on a side of the housing proximate the upper endand fluidly connected to the well.

Also disclosed herein are multi-stage acoustophoretic systems, such asthree- and four-stage acoustophoretic systems. These multi-stageacoustophoretic systems can incorporate a filter “train” comprisingdepth filters, sterile filters, centrifuges, and affinity chromatographycolumns to purify a cell culture by separating recombinant proteinstherefrom. In such systems, the frequency/power of the multi-dimensionalacoustic standing wave(s) generated in the system may be varied to senddifferent concentrations or different size materials to subsequentfiltration steps in the filter “train,” thereby improving the efficiencyof the clarification process by effectively managing the material thatis processed in each step in the filter “train.”

A multi-stage acoustophoretic system according to the present disclosureincludes three or more acoustophoretic devices fluidly connected to oneanother, each acoustophoretic device comprising: a flow chamber havingat least one inlet and at least one outlet; at least one ultrasonictransducer located on a wall of the flow chamber, the transducerincluding a piezoelectric material driven by a voltage signal to createa multi-dimensional standing wave in the flow chamber; and a reflectorlocated on the wall on the opposite side of the flow chamber from the atleast one ultrasonic transducer.

The acoustophoretic devices of the multi-stage acoustophoretic systemcan be fluidly connected to one another by tubing. The acoustophoreticdevices of the multi-stage acoustophoretic system can be physicallyconnected directly to one another, either one stage atop another, orside-by-side. The multi-stage acoustophoretic system can include a totalof four acoustophoretic devices.

Methods for continuously separating a second fluid or a particulate froma host fluid using a multi-stage acoustophoretic system are alsodisclosed. All of the multi-dimensional standing wave(s) created in eachacoustophoretic device of the multi-stage acoustophoretic system canhave different frequencies, or the same frequency, or can havefrequencies within the same order of magnitude.

These and other non-limiting characteristics are more particularlydescribed below.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawings, which arepresented for the purposes of illustrating the exemplary embodimentsdisclosed herein and not for the purposes of limiting the same.

FIG. 1 is an exterior perspective view of a basic acoustophoresis devicemade from three different modules: an ultrasonic transducer module, acollection well module, and an inlet/outlet module.

FIG. 2 is a perspective view of the ultrasonic transducer module ofFigure

FIG. 3 is a perspective view of an exemplary ultrasonic transducermodule in which an additional port is included between the ultrasonictransducer and the reflector.

FIG. 4 is a perspective view of the collection well module of FIG. 1.

FIG. 5 is a perspective view of the inlet/outlet module of FIG. 1. Here,the inlet port and the outlet port are located on a front wall of themodule.

FIG. 6 is a front view of the inlet/outlet module of FIG. 5.

FIG. 7 is a front view of an angled collection well module.

FIG. 8 is a front view of a second exemplary inlet/outlet module. Here,the inlet port and the outlet port are located at the upper end of thehousing.

FIG. 9 is a front view of an exemplary port module that functions as aninlet or an outlet, but not both at the same time. Put another way,fluid only flows in one direction through the port module.

FIG. 10 is a front view of a first exemplary two-way connector module.The flow channel in this connector module makes a 90° curve.

FIG. 11 is a front view of an exemplary three-way connector module. Thisconnector module has a total of three openings. Two openings are onopposite ends of the connector module. The third opening is located on aside of the connector module between the two ends.

FIG. 12 is a front view of a second exemplary two-way connector module.The flow channel in this connector module is straight. Two attachmentmembers are present, and both are of the same structure. Here, both arefemale members.

FIG. 13 is a front view of another exemplary two-way connector modulesimilar to FIG. 12, except that the attachment members are male members.

FIG. 14 is a perspective view of an ultrasonic transducer module thatincorporates a separation system formed from baffles.

FIG. 15 is a side view of the ultrasonic transducer module of FIG. 14.

FIG. 16 is a perspective view of the ultrasonic transducer module ofFIG. 14 joined to a collection well module having an angled lower end.

FIG. 17 is a side view of a first modular acoustophoretic system.

FIG. 18 is a side view of a second modular acoustophoretic system.

FIG. 19 is a side view of a third modular acoustophoretic system.

FIG. 20 is a side view of a fourth modular acoustophoretic system.

FIG. 21 is a side view of a fifth modular acoustophoretic system.

FIG. 22 is a side view of a sixth modular acoustophoretic system.

FIG. 23 is a side view of a seventh modular acoustophoretic system.

FIG. 24 is a side view of an eighth modular acoustophoretic system.

FIG. 25 is a side view of a ninth modular acoustophoretic system.

FIG. 26 is a side view of a tenth modular acoustophoretic system.

FIG. 27 is a side view of an eleventh modular acoustophoretic system.

FIG. 28 is a side view of a twelfth modular acoustophoretic system.

FIG. 29 is a side view of a thirteenth modular acoustophoretic system.

FIG. 30 is a cross-sectional diagram of a conventional ultrasonictransducer.

FIG. 31 is a cross-sectional diagram of an ultrasonic transducer of thepresent disclosure. An air gap is present within the transducer, and nobacking layer or wear plate is present.

FIG. 32 is a cross-sectional diagram of an ultrasonic transducer of thepresent disclosure. An air gap is present within the transducer, and abacking layer and wear plate are present.

FIG. 33 is a graph of electrical impedance amplitude versus frequencyfor a square transducer driven at different frequencies.

FIG. 34A illustrates the trapping line configurations for seven of thepeak amplitudes of FIG. 33 from the direction orthogonal to fluid flow.

FIG. 34B is a perspective view illustrating the separator. The fluidflow direction and the trapping lines are shown.

FIG. 34C is a view from the fluid inlet along the fluid flow direction(arrow 114) of FIG. 34B, showing the trapping nodes of the standing wavewhere particles would be captured.

FIG. 34D is a view taken through the transducers face at the trappingline configurations, along arrow 116 as shown in FIG. 34B.

FIG. 35 is a graph showing the relationship of the acoustic radiationforce, buoyancy force, and Stokes' drag force to particle size. Thehorizontal axis is in microns (μm) and the vertical axis is in Newtons(N).

FIG. 36 illustrates an exemplary embodiment of a multi-stageacoustophoretic system according to the present disclosure. Theacoustophoretic system includes a total of four stages fluidly connectedto one another by tubing.

FIG. 37A illustrates an exemplary embodiment of four acoustophoreticdevices/stages physically connected to one another for use in amulti-stage acoustophoretic system according to the present disclosure.

FIG. 37B illustrates an isolated view of one of the acoustophoreticdevices/stages of FIG. 37A.

FIG. 37C is a cross-sectional diagram of one of the acoustophoreticdevices/stages of FIG. 37A. The device includes opposing flow dumpdiffuser inlets generating flow symmetry and more uniform velocities.

FIG. 38 is a performance chart showing the pressure drop for a system ofthe present disclosure having three acoustophoretic devices in series ina first experiment. The y-axis is pressure drop in psig, and runs from 0to 30 in intervals of 5. The x-axis is volumetric throughput capacity inliters per square meter, and runs from 0 to 140 in intervals of 20.

FIG. 39 is a set of two graphs showing the performance of a secondexperimental system. The bottom graph has a y-axis of percent reduction,and runs from 0% to 100% in intervals of 10%. The x-axis is testduration in minutes, and runs from 0 to 80 in intervals of 10. The topgraph has a y-axis of percent reduction, and is logarithmic, with valuesof 0, 90%, and 99.0%.

FIG. 40 is a set of graphs showing the performance of a thirdexperimental system. The bottom graph has a y-axis of percent reduction,and runs from 0% to 100% in intervals of 10%. The x-axis is testduration in minutes, and runs from 0 to 80 in intervals of 10. The topgraph has a y-axis of percent reduction, and is logarithmic, with valuesof 0, 90%, and 99.0%.

FIG. 41 is a graph showing the performance of multiple experimental testsystems. The y-axis is total cell density (TCD) reduction percentage inincrements of 10% from 0% to 100%. Along the x-axis are five distincttests. The first bar represents a three-stage system with all threestages operated at 40V (test 1). The second bar represents a three-stagesystem with all three stages operated at 50V (test 2). The third barrepresents a three-stage system with all three stages operated at 60V(test 3). The fourth bar represents a three-stage system with the firststage operated at 60V, the second stage operated at 50V, and the thirdstage operated at 40V (test 4). The fifth bar represents a four-stagesystem with all four stages operated at 50V (test 5).

FIG. 42 is another graph showing the performance of each stage of FIG.41. The y-axis is cumulative TCD reduction percentage per stage at 30minutes in increments of 10% from 0% to 100%. Along the x-axis are thethree stages of each system. The first series of bars represents thecumulative % reduction after the first stage of each system. The secondseries of bars represents the cumulative % reduction after the secondstage of each system. The third series of bars represents the cumulative% reduction after the third stage of each system. Within each series ofbars, the leftmost bar is test 1, the second bar from the left is test2, and the second bar from the right is test 3, and the rightmost bar istest 4.

FIG. 43 is another graph showing the performance of each stage of thetest systems of FIG. 41. The y-axis is cumulative TCD reductionpercentage per stage at 30 minutes in increments of 10% from 0% to 100%.Along the x-axis are the three stages of each system. The first seriesof bars represents the cumulative % reduction after the first stage ofeach system. The second series of bars represents the cumulative %reduction after the second stage of each system. The third series ofbars represents the cumulative % reduction after the third stage of eachsystem. Within each series of bars, the leftmost bar is test 1, thesecond bar from the left is test 2, the middle bar is test 3, the secondbar from the right is test 4, and the rightmost bar is test 5. For stage4, the only bar shown is for test 5.

FIG. 44 is another graph showing the performance of the five testsystems of FIG. 41. The y-axis is total turbidity reduction percentagein increments of 10% from 0% to 100%. Along the x-axis are five distincttests. The first bar represents a three-stage system with all threestages operated at 40V (test 1). The second bar represents a three-stagesystem with all three stages operated at 50V (test 2). The third barrepresents a three-stage system with all three stages operated at 60V(test 3). The fourth bar represents a three-stage system with the firststage operated at 60V, the second stage operated at 50V, and the thirdstage operated at 40V (test 4). The fifth bar represents a four-stagesystem with all four stages operated at 50V (test 5).

FIG. 45 is a graph showing the performance or a volume throughput (VT)of a fifth experimental system. The top line along the right side of thegraph represents the D0HC filter pressure drop. The dotted lineextending from the D0HC pressure drop line represents the D0HC filterturbidity. The bottom line along the right side of the graph representsthe X0HC filter pressure drop. The dotted line extending from the XOHCpressure drop line represents the X0HC filter turbidity.

FIG. 46 is another graph showing the performance or volume throughput(VT) of the fifth experimental system.

FIG. 47 is a graph showing the performance of a sixth experimentalsystem.

FIG. 48 is another graph showing the performance of the sixthexperimental system utilizing depth flow filtration (DFF) and volumethroughput (VT) as a performance indicator. The y-axis is volumetricthroughput in L/m², and runs from 0 to 250 in intervals of 50. The barlabeled “harvest feed” is the throughput through a two-stage depthfilter. The bars labeled “AWS (3-stage)” are the throughput throughfilters obtained from two different manufacturers after filtrationthrough a 3-stage system. The bars labeled “AWS (4-stage)” are thethroughput through filters obtained from four different manufacturersafter filtration through a 4-stage system.

FIG. 49 illustrates an exemplary setup of a three-stage acoustophoreticsystem used as the experimental system for some performance testing ofthe present disclosure.

FIG. 50 illustrates an exemplary setup of a four-stage acoustophoreticsystem used as the experimental system for much of the performancetesting of the present disclosure.

FIG. 51 is a graph showing turbidity reduction vs feed PCM for thesystem of FIG. 50.

FIG. 52 is a graph showing percent reduction vs feed flow rate for threedifferent factors (TCD, turbidity, PCM) for the system of FIG. 50.

FIG. 53 is a graph showing turbidity reduction vs flow ratio for thesystem of FIG. 50.

FIG. 54 is a graph showing PCM vs solids flow ratio for the system ofFIG. 50.

FIG. 55 is a graph showing turbidity reduction vs flow ratio for thesystem of FIG. 50.

FIG. 56 is a bar graph showing solids PCM vs flow ratio for the systemof FIG. 50.

FIG. 57 is another graph showing the clarification performance for thesystem of FIG. 50. For each set of bars, the leftmost bar is Q_(F)=1.0x,3-stage; the middle bar Q_(F)=0.6x, 3-stage; and the right bar isQ_(F)=1.0x, 4-stage.

FIG. 58 is a graph showing % reduction vs feed PCM for the system ofFIG. 50.

FIG. 59 is a graph showing % reduction vs feed cell viability for thesystem of FIG. 50.

FIG. 60 is a graph showing the performance of a seventh experimentalsystem.

FIG. 61 is another graph showing the performance of the seventhexperimental system. The performance of each stage is shown over time,and shows that the performance does not deteriorate.

FIG. 62 is another graph showing the performance of the seventhexperimental system. This compares the depth filter area needed for adepth filter versus the equivalent area for an acoustophoretic filter ofthe same capacity.

FIG. 63 is another graph showing the performance of the seventhexperimental system.

DETAILED DESCRIPTION

The present disclosure may be understood more readily by reference tothe following detailed description of desired embodiments and theexamples included therein. In the following specification and the claimswhich follow, reference will be made to a number of terms which shall bedefined to have the following meanings.

Although specific terms are used in the following description for thesake of clarity, these terms are intended to refer only to theparticular structure of the embodiments selected for illustration in thedrawings, and are not intended to define or limit the scope of thedisclosure. In the drawings and the following description below, it isto be understood that like numeric designations refer to components oflike function. Furthermore, it should be understood that the drawingsare not to scale.

In the figures, interior surfaces are designated by dashed lines incross-sectional views, unless otherwise noted.

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise.

As used in the specification and in the claims, the term “comprising”may include the embodiments “consisting of” and “consisting essentiallyof.” The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that require thepresence of the named components/steps and permit the presence of othercomponents/steps. However, such description should be construed as alsodescribing compositions or processes as “consisting of” and “consistingessentially of” the enumerated components/steps, which allows thepresence of only the named components/steps, along with any impuritiesthat might result therefrom, and excludes other components/steps.

Numerical values should be understood to include numerical values whichare the same when reduced to the same number of significant figures andnumerical values which differ from the stated value by less than theexperimental error of conventional measurement technique of the typedescribed in the present application to determine the value.

All ranges disclosed herein are inclusive of the recited endpoint andindependently combinable (for example, the range of “from 2 grams to 10grams” is inclusive of the endpoints, 2 grams and 10 grams, and all theintermediate values).

A value modified by a term or terms, such as “about” and“substantially,” may not be limited to the precise value specified. Theapproximating language may correspond to the precision of an instrumentfor measuring the value. The modifier “about” should also be consideredas disclosing the range defined by the absolute values of the twoendpoints. For example, the expression “from about 2 to about 4” alsodiscloses the range “from 2 to 4.”

It should be noted that many of the terms used herein are relativeterms. For example, the terms “upper” and “lower” are relative to eachother in location, i.e. an upper component is located at a higherelevation than a lower component in a given orientation, but these termscan change if the device is flipped. The terms “inlet” and “outlet” arerelative to a fluid flowing through them with respect to a givenstructure, e.g. a fluid flows through the inlet into the structure andflows through the outlet out of the structure. The terms “upstream” and“downstream” are relative to the direction in which a fluid flowsthrough various components, i.e. the flow fluids through an upstreamcomponent prior to flowing through the downstream component. It shouldbe noted that in a loop, a first component can be described as beingboth upstream of and downstream of a second component.

The terms “horizontal” and “vertical” are used to indicate directionrelative to an absolute reference, i.e. ground level. The terms“upwards” and “downwards” are also relative to an absolute reference; anupwards flow is always against the gravity of the earth.

The present application refers to “the same order of magnitude.” Twonumbers are of the same order of magnitude if the quotient of the largernumber divided by the smaller number is a value of at least 1 and lessthan 10.

The present application also refers to an “acute” angle. For purposes ofthe present disclosure, the term “acute” refers to an angle between 0°and 90°, exclusive of 0° and 90°.

The acoustophoretic separation technology of the present disclosureemploys ultrasonic standing waves to trap, i.e., hold stationary,secondary phase particles in a host fluid stream. This is an importantdistinction from previous approaches where particle trajectories weremerely altered by the effect of the acoustic radiation force. Thescattering of the acoustic field off the particles results in a threedimensional acoustic radiation force, which acts as a three-dimensionaltrapping field. The acoustic radiation force is proportional to theparticle volume (e.g. the cube of the radius) when the particle is smallrelative to the wavelength. It is proportional to frequency and theacoustic contrast factor. It also scales with acoustic energy (e.g. thesquare of the acoustic pressure amplitude). For harmonic excitation, thesinusoidal spatial variation of the force is what drives the particlesto the stable positions within the standing waves. When the acousticradiation force exerted on the particles is stronger than the combinedeffect of fluid drag force and buoyancy and gravitational force, theparticle is trapped within the acoustic standing wave field. Thisresults in concentration, agglomeration and/or coalescence of thetrapped particles. Additionally, secondary inter-particle forces, suchas Bjerkness forces, aid in particle agglomeration.Heavier-than-the-host-fluid (i.e. denser than the host fluid) particlesare separated through enhanced gravitational settling.

One specific application for the acoustophoresis device is in theprocessing of bioreactor materials. It is important to be able to filterall of the cells and cell debris from the expressed materials that arein the fluid stream. The expressed materials are composed ofbiomolecules such as recombinant proteins or monoclonal antibodies, andare the desired product to be recovered. Through the use ofacoustophoresis, the separation of the cells and cell debris is veryefficient and leads to very little loss of the expressed materials. Thisis an improvement over current filtration processes (depth filtration,tangential flow filtration, centrifugation), which show limitedefficiencies at high cell densities, so that the loss of the expressedmaterials in the filter beds themselves can be up to 5% of the materialsproduced by the bioreactor. The use of mammalian cell cultures includingChinese hamster ovary (CHO), NS0 hybridoma cells, baby hamster kidney(BHK) cells, and human cells has proven to be a very efficacious way ofproducing/expressing the recombinant proteins and monoclonal antibodiesrequired of today's pharmaceuticals. The filtration of the mammaliancells and the mammalian cell debris through acoustophoresis aids ingreatly increasing the yield of the bioreactor.

In this regard, the contrast factor is the difference between thecompressibility and density of the particles and the fluid itself. Theseproperties are characteristic of the particles and the fluid themselves.Most cell types present a higher density and lower compressibility thanthe medium in which they are suspended, so that the acoustic contrastfactor between the cells and the medium has a positive value. As aresult, the axial acoustic radiation force (ARF) drives the cells, witha positive contrast factor, to the pressure nodal planes, whereas cellsor other particles with a negative contrast factor are driven to thepressure anti-nodal planes. The radial or lateral component of theacoustic radiation force helps trap the cells. The radial or lateralcomponent of the ARF is larger than the combined effect of fluid dragforce and gravitational force.

As the cells agglomerate at the nodes of the standing wave, there isalso a physical scrubbing of the cell culture media that occurs wherebymore cells are trapped as they come in contact with the cells that arealready held within the standing wave. This generally separates thecells from the cell culture media. The expressed biomolecules remain inthe nutrient fluid stream (i.e. cell culture medium).

Desirably, the ultrasonic transducer(s) generate a three-dimensional ormulti-dimensional acoustic standing wave in the fluid that exerts alateral force on the suspended particles to accompany the axial force soas to increase the particle trapping capabilities of the standing wave.Typical results published in literature state that the lateral force istwo orders of magnitude smaller than the axial force. In contrast, thetechnology disclosed in this application provides for a lateral force tobe of the same order of magnitude as the axial force.

It is also possible to drive multiple ultrasonic transducers witharbitrary phasing. In other words, the multiple transducers may work toseparate materials in a fluid stream while being out of phase with eachother. Alternatively, a single ultrasonic transducer that has beendivided into an ordered array may also be operated such that somecomponents of the array will be out of phase with other components ofthe array.

Three-dimensional (3-D) or multi-dimensional acoustic standing waves aregenerated from one or more piezoelectric transducers, where thetransducers are electrically or mechanically excited such that they movein a multi-excitation mode. The types of waves thus generated can becharacterized as composite waves, with displacement profiles that aresimilar to leaky symmetric (also referred to as compressional orextensional) Lamb waves. The waves are leaky because they radiate intothe water layer, which result in the generation of the acoustic standingwaves in the water layer. Symmetric Lamb waves have displacementprofiles that are symmetric with respect to the neutral axis of thepiezoelectric element, which causes multiple standing waves to begenerated in a 3-D space. Through this manner of wave generation, ahigher lateral trapping force is generated than if the piezoelectrictransducer is excited in a “piston” mode where only a single, planarstanding wave is generated. Thus, with the same input power to apiezoelectric transducer, the 3-D or multi-dimensional acoustic standingwaves can have a higher lateral trapping force which may be up to andbeyond 10 times stronger than a single acoustic standing wave generatedin piston mode.

It may be necessary, at times, due to acoustic streaming, to modulatethe frequency or voltage amplitude of the standing wave. This may bedone by amplitude modulation and/or by frequency modulation. The dutycycle of the propagation of the standing wave may also be utilized toachieve certain results for trapping of materials. In other words, theacoustic beam may be turned on and shut off at different frequencies toachieve desired results.

The present disclosure relates to acoustophoresis devices that are madeof modular components, and to kits of such modules. The modules includeattachment members that are used to cooperatively engage or fit withother modules and can then be reversibly separated. The kits and modulespermit the user to make different configurations of acoustophoresisdevices as needed to provide for improved settling and improvedseparation of particles from fluid. Briefly, particles that aresuspended in a host fluid can be subjected to multiple transducersgenerating multiple standing waves in different areas of the separationdevice to induce separation from the fluid itself. Improved fluiddynamics can also be provided using the modular components, increasingseparation of particles from fluid. For example, the fluid stream can bechanneled into two or more streams, or the fluid flow can proceed atvarious angles from 1° up to 90° normal to a base plane.

The use of multiple standing waves from multiple ultrasonic transducersallows for multiple separation stages. For example, in a flow path thatruns past two ultrasonic transducers, the first transducer (and itsstanding wave) will collect a certain amount of the particles, and thesecond transducer (and its standing wave) will collect additionalparticles that the first transducer was not able to hold. Thisconstruction can be useful where the particle/fluid ratio is high (i.e.large volume of particles), and the separation capacity of the firsttransducer is reached. This construction can also be useful forparticles that have a bimodal or greater size distribution, where eachtransducer can be optimized to capture particles within a certain sizerange.

FIG. 1 is an exterior perspective view of a basic acoustophoresis devicethat can be used for the purposes described above. This basicacoustophoresis device 100 is formed from a kit that includes anultrasonic transducer module 200, a collection well module 300, and aninlet/outlet module 400. As seen here, the three modules are reversiblyinterlocked together to form one or more flow paths 102 into which afluid/particle mixture can be processed to separate the particles fromthe fluids or to further concentrate the particles within the mixture,and to recover the particles/concentrated mixture.

Briefly, in FIG. 1 the inlet/outlet module 400 contains an inlet port432 and an outlet port 434 for the flow path. A fluid/particle mixtureis pumped in through the inlet port 432. The mixture flows downwards viagravity through the ultrasonic transducer module 200, where theparticles are trapped and held by the ultrasonic standing wave. As fluidcontinues to be pumped into the flow path, eventually the collectionwell module 300 and the ultrasonic transducer module 200 are filled withfluid, and the fluid pressure rises high enough that fluid will flow outthrough the outlet port 434 at the top of the device. The particleswithin the ultrasonic standing wave collect or agglomerate, andeventually grow to a size where gravity overcomes the acoustic force ofthe standing wave, and the particle aggregates then fall/sink into thecollection well module 300. The collection well module includes a well330 that tapers downwards in cross-sectional size to a vertex 334. Adrain line 340 connects the vertex 334 to a port 342 where theconcentrated particles can be drawn out of the well.

FIG. 2 is a perspective view of the ultrasonic transducer module ofFIG. 1. The ultrasonic transducer module 200 includes a housing 202having a first end 204 and a second end 206 which are located atopposite ends of the housing. Here, the housing is in the shape of acube having four side walls 210, 212 (third and fourth walls notvisible), 216, a first wall 220, and a second wall 222. However, theexterior shape of the module is not particularly relevant, and could befor example cylindrical. The first end and the second end of the housingcan be considered as defining a z-axis. The four opposing side walls canbe considered as corresponding to opposite directions along the x-y axesof the housing.

A flow channel 230 is defined between the first end 204 and the secondend 206 of the housing. Put another way, an opening 232, 234 is presentin both the first wall and the second wall, and a bore joins the twoopenings together, such that fluid can flow through the housing frombetween the first end and the second end. As illustrated here, the borehas a rectangular (e.g. square) cross-section. An ultrasonic transducer240 is located on one side of the housing, and the reflector 242 islocated on the side of the housing opposite the ultrasonic transducer.It should be noted that the ultrasonic transducer is directly adjacentto the flow channel, and would be directly exposed to any fluid passingthrough the flow channel. The reflector is solid or flexible, and can bemade of a high acoustic impedance material such as steel or tungsten,providing good reflection.

A first attachment member 260 is disposed at the first end 204 of thehousing, i.e. on the first wall 220 of the housing. A second attachmentmember 262 is disposed at the second end 206 of the housing, i.e. on thesecond wall 222 of the housing. These attachment members are intended topermit the module to be reversibly joined with other modules and form awater-tight seal. As illustrated here, the second attachment member iscomplementary to the first attachment member. The second attachmentmember is a male member (e.g. a tongue), and the first attachment memberis a female member (e.g. a hole). An o-ring (not shown) is present onthe second attachment member to ensure the seal. The first end 204 ofthe housing also includes four tenons 270, one located at each corner,and the second end 206 includes four mortises 272, again located at eachcorner. The depicted attachment members are intended to be press-fittedtogether. Of course, other reversible attachment means are contemplated,for example attachment members that include internal or externalthreads, so that modules are screwed together. The attachment memberscould also be reversed in location (e.g. the first attachment member ismale, and the second attachment member is female). These attachmentmembers can also be described as surrounding the openings in the firstwall and second wall.

In some embodiments of the ultrasonic transducer module, as seen in FIG.3, an additional port 274 can be included on a side 214 of the housingbetween the transducer 240 and the reflector 242. This may be useful forinjecting fluid into the particles trapped by the acoustic standingwave, or for collecting particles directly from the acoustic standingwave.

FIG. 4 is a perspective view of the collection well module of FIG. 1.The collection well module 300 includes a housing 302 having an upperend 304 and a lower end 306. As illustrated, the housing is in the shapeof a cube having four sides, an upper wall 320, and a lower wall 322.More generally, the upper end and the lower end of the housing arelocated at opposite ends of the housing, and can be considered asdefining a z-axis. The housing also has four opposing sides 310, 312,314, 316, which can be considered as corresponding to oppositedirections along the x-y axes of the housing. Again, the exterior shapeof the module is not particularly relevant. However, it is noted thatthe collection well module is usually located at the bottom of theoverall acoustophoretic device, and so the lower wall usually provides abase for the device and should be flat.

An inlet 332 is present in the upper end/upper wall of the housing, andis intended to receive particles and fluid. As illustrated here, theinlet has a rectangular (e.g. square) cross-section. A well 330 ispresent in the housing, which tapers downwards in cross-sectional areafrom the inlet 332 to a vertex 334. The inlet forms one end of the well,and the vertex forms the other end of the well. A drain line 340connects the vertex 334 to a port 342 on a side of the housing, fromwhich a concentrated particle/fluid mixture can flow out of the well 330to the port. It is noted that because the lower wall acts as a base, theport is located on one of the four opposing sides of the housing. Itshould be noted that this collection well module has only one inlet 332,i.e. does not have two or more inlets. Also, the well is depicted herewith the inlet 332 and the vertex 334 being concentric, i.e. when viewedfrom the top, the vertex is in the center of the inlet. However, thisconcentricity is not required. For example, the vertex could be skewedto the side to minimize the length of the drain line.

An attachment member 360 is disposed at the upper end 304 of thehousing, i.e. on the upper wall of the housing, and again is intended topermit the module to be reversibly joined with other modules and form awater-tight seal. As illustrated here, the attachment member is a femalemember (e.g. a hole). In addition, the upper end of the housing alsoincludes four tenons 370 at each corner. Here, the attachment member ofthe collection well module is complementary to the lower attachmentmember of the ultrasonic transducer module. Again, the attachment membercan also be described as surrounding the inlet.

FIG. 5 is a perspective view of the inlet/outlet module of FIG. 1. FIG.6 is a front view of the inlet/outlet module. In this regard, theinlet/outlet module 400 is adapted to both introduce a particle/fluidmixture into the flow path, as well as to expel/remove fluid from theflow path. The inlet/outlet module 400 includes a housing 402 having anupper end 404 and a lower end 406. As illustrated, the housing generallyhas an upper wall 420, a lower wall 422, and at least one side wall 410extending between them. The upper end and the lower end of the housingare located at opposite ends of the housing, and can be considered asdefining a z-axis. The housing also has four opposing sides 410, 412,414 (fourth wall not visible), which can be considered as correspondingto opposite directions along the x-y axes of the housing. Again, theexterior shape of the module is not particularly relevant.

The inlet/outlet module includes an inlet port 432 and an outlet port434, which are illustrated here as being spaced apart from each other ona common side of the housing (i.e. front wall 412). An opening 436 ispresent at the lower end 406 of the housing (i.e. in the lower wall). Aflow channel 430 is defined by the inlet port 432, the outlet port 434,and the opening 436. The inlet port and the outlet port are located at afirst end of the flow channel, and the opening is located at the secondend of the flow channel.

As best seen in FIG. 6, a wall 440 is located in the flow channel 430between the inlet port 432 and the outlet port 434. Due to the presenceof the wall, as explained above in the discussion of FIG. 1, fluid flowsfrom the inlet port through the opening and then to the outlet port. Thewall essentially divides the flow channel into two separatesub-channels, the ends of one sub-channel 480 being identified by theinlet port 432 and the opening 436, and the ends of the othersub-channel 482 being identified by the outlet port 434 and the opening436. The cross-sectional area of the flow channel for the inlet port canbe smaller than, equal to, or greater than the cross-sectional area ofthe flow channel for the outlet port. As illustrated in FIG. 6, the wallis placed so that the cross-sectional area 441 of the flow channel forthe inlet port is smaller than the cross-sectional area 443 of the flowchannel for the outlet port.

Also visible in FIG. 6 is a first retainer wall 442 adjacent the inletport and a second retainer wall 444 adjacent the outlet port. As seenhere, the inlet port 432 and the outlet port 434 are located relativelyclose to the middle of the front wall, and are spaced apart from theupper end 404 of the housing. Incoming fluid must flow towards the upperend 404 and then over the first retainer wall 442 before exiting throughthe opening 436. Similarly, fluid coming back from the ultrasonictransducer module must flow from the opening 436 over the secondretainer wall 444 before exiting through the outlet port 434. Thisconstruction provides a means by which the turbulence of incoming fluidcan be reduced, so that the particles trapped in the acoustic standingwave in the ultrasonic transducer module are not disrupted or washed outof the standing wave before aggregating to a sufficient size.

In some embodiments such as the one depicted here, the wall 440 extendsout of the opening 436 at the lower end of the housing. This helpsensure that the incoming particle/fluid mixture passes through theultrasonic transducer module before fluid exits the flow path (of theoverall acoustophoresis device) through the outlet port.

As also depicted here, in some embodiments, the wall 440 is spaced apartfrom the upper end 404/upper wall 420 of the housing. This gap 446 formsand acts as a pressure relief passage between the inlet port 432 and theoutlet port 434, for example in case the flow path is inadvertentlyblocked.

Continuing with FIG. 6, an attachment member 460 is disposed at thelower end 406 of the housing, i.e. on the lower wall of the housing, andagain is intended to permit the module to be reversibly joined withother modules and form a water-tight seal. As illustrated here, theattachment member is a male member (e.g. a tongue). An o-ring (notshown) is used on the attachment member to ensure the seal. Fourmortises 472 are also present, one at each corner on the lower end ofthe housing.

Besides the three modules described above, additional modules arecontemplated that can be used to form an acoustophoretic system asdescribed above. These modules include an angled collection well module500, another inlet/outlet module 600, a port module 700, variousconnector modules 800, and transducer modules combined with an improvedseparation system 900. These various modules will now be described.

FIG. 7 is a front view of an angled collection well module 500. Theangled collection well module includes a housing 502 having an upper end504 and a lower end 506. The exterior shape of the module is notparticularly relevant. More generally, the upper end and the lower endof the housing are located at opposite ends of the housing, and can beconsidered as defining a z-axis. The lower end 506 defines a base of thehousing.

This module has a first opening 580 and a second opening 582. At leastone of the openings is located at an acute angle relative to the base ofthe housing. This is indicated on FIG. 7 with the first opening havingan angle A (dotted line). The first opening 580 can be described asbeing located on a side 510 of the housing. Here, the second opening 582is located opposite the base of the housing, i.e. on the upper end 504of the housing. However, it is also contemplated that the second openingcould also be located at an acute angle relative to the base of thehousing.

The first opening 580 and the second opening 582 both lead into a commonwell 530 that tapers downwards in cross-sectional area from the openingsto a vertex 534 (interior surface shown in dashed lines). A drain line540 connects the vertex 534 to a port 542 on a side of the housing, fromwhich a concentrated particle/fluid mixture can drain from the well 530to the port 542 and out of the collection module. Because the lower wallacts as a base, the port is located on a side of the housing. Inparticular embodiments, the angled collection well module has only twoopenings 580, 582. In other embodiments, the angled collection wellmodule has at least two openings located at an acute angle relative tothe base of the housing, with all openings leading into the common well530.

A first attachment member 560 is located at the first opening 580. Asecond attachment member 562 is located at the second opening 582. Eachattachment member can also be described as surrounding the opening. Theattachment members are intended to permit the module to be reversiblyjoined with other modules and form a water-tight seal. As illustratedhere, the first attachment member is a male member (e.g. a tongue), andthe second attachment member is a female member (e.g. a hole). An o-ring(not shown) is present on the first attachment member to ensure theseal. In addition, the second opening 582 also includes four tenons 570,one located at each corner. The first opening 580 also includes fourmortises 572, again located at each corner. In particular embodiments,the first attachment member is complementary to the second attachmentmember, and they are also adapted to engage and interlock with theultrasonic transducer module.

FIG. 8 is a front view of an alternative embodiment of the inlet/outletmodule. Similar to the module of FIG. 5, the inlet/outlet module 600includes a housing 602 having an upper end 604 and a lower end 606 whichare located at opposite ends of the housing. The housing generally has alower wall 622. An opening 636 is present at the lower end of thehousing (i.e. in the lower wall, interior surface denoted by dashedlines). The inlet/outlet module also includes an inlet port 632 and anoutlet port 634. A flow channel 630 is defined by the inlet port 632,the outlet port 634, and the opening 636. In this embodiment, the inletport 632 and the outlet port 634 are located at the upper end 604 of thehousing. The inlet port and the outlet port are located at a first endof the flow channel, and the opening is located at the second end of theflow channel. The flow channel here is in the shape of two tubes 680,682, each tube acting as a sub-channel. One tube 680 runs between theinlet port 632 and the opening 636. The other tube 682 runs between theoutlet port 634 and the opening 636. Again, the cross-sectional area 641of the tube for the inlet port can be smaller than, equal to, or greaterthan the cross-sectional area 643 of the tube for the outlet port. Asillustrated here, the cross-sectional areas of the two tubes are equal.An attachment member 660 is present at the lower end 606, hereillustrated as a male member (e.g. tongue).

FIG. 9 is a front view of a port module 700 that can be combined withthe other modules of the present disclosure. The port module 700includes a housing 702 having an upper end 704 and a lower end 706 whichare located at opposite ends of the housing. As illustrated here, theport module is in the shape of a cone with a plate 722 on the lower end.Again, though, the exterior shape is not particularly relevant. A flowchannel 730 is defined between the upper end 704 and the lower end 706of the housing (indicated by dashed lines). Put another way, an opening732, 734 is present at both the upper end and the lower end, and a borejoins the two openings together, such that fluid can flow through thehousing from between the upper end and the lower end. Here, the flowchannel is in the shape of a cone, which acts as a diffuser. In contrastto the inlet/outlet module of FIG. 5 and FIG. 8, the flow channel of theport module allows fluid flow in only one direction. The port module canfunction as an inlet or an outlet, but not both at the same time. Putanother way, the flow channel is not made of sub-channels.

The port module 700 also includes an attachment member 760 at the lowerend of the housing for joining the port module to other modules andforming a water-tight seal. As illustrated here, the attachment memberis a male member (e.g. a tongue), with an o-ring (not shown) on theattachment member to ensure the seal. In addition, four mortises 772 arepresent, one at each corner on the lower end of the housing.

FIG. 10 is a front view of an exemplary two-way connector module 800.The connector module 800 includes a housing 802 having an upper end 804and a lower end 806 which are located at opposite ends of the housing.Again, generally, the upper end and the lower end of the housing can beconsidered as defining a z-axis. The housing also has four opposingsides 810, 812, 814 (fourth side not visible), which can be consideredas corresponding to opposite directions along the x-y axes of thehousing.

This module has a first opening 832 and a second opening 834. Oneopening is present in the upper end 804 of the housing, and the otheropening is present in a side 810 of the housing. A flow channel 830 isdefined between the two openings, with a bore joining the two openingstogether to permit fluid to flow through the housing between the twoopenings (indicated by dashed lines). As seen here, the flow channel iscurved about 90°.

A first attachment member 860 is located at the first opening 832. Asecond attachment member 862 is located at the second opening 834. Eachattachment member can also be described as surrounding the opening. Theattachment members are intended to permit the connector module to bereversibly joined with other modules and form a water-tight seal. Asillustrated here, the first attachment member 860 is a male member (e.g.a tongue), and the second attachment member 862 is a female member (e.g.a hole). An o-ring (not shown) is present on the first attachment memberto ensure the seal. In addition, the second opening 834 also includesfour tenons 870, one located at each corner. The first opening 832 alsoincludes four mortises 872, again located at each corner. In particularembodiments, the first attachment member 860 is complementary to thesecond attachment member 862, and they are also adapted to engage andinterlock with the ultrasonic transducer module 200.

The connector 800 of FIG. 10 has only two openings. FIG. 11 is a frontview of a three-way connector module 892. This connector module has atotal of three openings. The structure of this connector module is verysimilar to the structure 800 of FIG. 10. The only addition is theinclusion of the third opening 836 in the lower end 806 of the housing802. A third attachment member 864 is also present at the lower end 806of the housing. As a result, the flow channel 830 is T-shaped,permitting flow between any combination of the three openings 832, 834,836. In specific embodiments, the third attachment member 864 iscomplementary to the first attachment member 860, and is notcomplementary to the second attachment member 862. In other embodiments,the third attachment member 864 is complementary to the secondattachment member 862, and is not complementary to the first attachmentmember 860.

FIG. 12 is a front view of another two-way connector module 894. Thisconnector module includes a housing 802 having an upper end 804 and alower end 806 which are located at opposite ends of the housing. Themodule also has a first opening 832 and a second opening 834. Oneopening 832 is present in the upper end 804 of the housing, and theother opening 834 is present in the lower end 806 of the housing. A flowchannel 830 is defined between the two openings (indicated with dashedlines), with a bore joining the two openings together to permit fluid toflow through the housing between the two openings.

A first attachment member 860 is located at the first opening 832. Asecond attachment member 862 is located at the second opening 834. Eachattachment member can also be described as surrounding the opening. Thetwo attachment members are of the same type and structure. Here, the twoattachment members 860, 862 are female (e.g. a hole). In addition, eachopening also includes four tenons 870, one located at each corner.

FIG. 13 is a perspective view of a third two-way connector module 896.This embodiment is similar to the connector module 894 of FIG. 12,except the two attachment members 860, 862 are male (e.g. a tongue). Inaddition, each opening also includes four mortises 872, again located ateach corner.

The connector modules of FIG. 12 and FIG. 13 are intended to permit theorientation of a given opening on a different module to be reversed. Theutility of such connectors will be shown later.

FIG. 14 is a perspective view of an ultrasonic transducer module 900that incorporates a separation system formed from baffles. FIG. 15 is aside view (y-z plane) of the ultrasonic transducer module of FIG. 14.

This ultrasonic transducer module 900 has many of the same components asthe ultrasonic transducer module of FIG. 2, including the housing 902with the first end 904, second end 906, and four side walls 910, 912,914, 916. A primary flow channel 930 is defined between the first endand the second end of the housing, as represented by circular openings932, 934. The first end 904 and the second end 906 of the housing can beconsidered as defining a z-axis. The sides of the housing on which theultrasonic transducer (not shown) and the reflector (not shown) would belocated are represented by square openings 936, 938, and can beconsidered as defining a y-axis.

In the ultrasonic transducer of FIG. 14, an angled extension 940 extendsfrom one of the sides 910 between the ultrasonic transducer 936 and thereflector 938. A secondary flow channel 942 is present within the angledextension 940, the secondary flow channel connecting to the primary flowchannel 930 between the first end 904 and the second end 906 of themodule. A set of baffles 944 is located within the secondary flowchannel 942. The baffles are flat plates. The baffles 944 lead to athird opening 946 at the distal end 948 of the angled extension940/secondary flow channel 942. A third attachment member 964 isdisposed at the distal end 948 of the angled extension 940. Asillustrated here, the first attachment member 960 and the secondattachment member 962 are both female members (e.g. a hole), and thethird attachment member 964 is a male member (e.g. a tongue).

In one mode of operation illustrated in FIG. 16, it is contemplated thatthe ultrasonic transducer module of FIG. 14 will be oriented such thatthe angled extension 940 acts as a base. The acoustic standing wavefield will trap particles and cause aggregation until the particleaggregate is heavy enough for gravity to cause the aggregate to falldownwards and out of the acoustic standing wave field. The aggregatethen falls down onto the baffles 944, which acts as a collection surfaceto guide the aggregate to the collection well module.

In another mode of operation, it is contemplated that the ultrasonictransducer module of FIG. 14 will be oriented such that the angledextension 940 points upwards, i.e. against the flow of gravity (theupwards direction indicated by arrow 905 in FIG. 15). Fluid flows pastthe ultrasonic transducer 936, then upwards through the angled extension940. As the fluid flows upwards over and through the baffles, particlesthat escape the ultrasonic transducer will contact the baffles 944. Thebaffles will retard the particles, and can cause them to fall downwardsback towards the acoustic standing wave field generated by theultrasonic transducer 936, or towards a collection well device (notdepicted).

FIG. 16 is a perspective view of the ultrasonic transducer module 900 ofFIG. 13 joined to a collection well module 300. This collection wellmodule is another variation of the collection well module 300 previouslydescribed in FIG. 4. This module also has four side walls, an upper wall320, a lower wall 322, a well 330, and a port 342. Notably, the lowerwall 322 is angled, rather than parallel to the upper wall as in FIG. 4.The angle of the lower wall is the same as the angle of the angledextension. This provides a flat base for supporting the ultrasonictransducer module.

The various modules discussed above can be made from any suitablematerial. Such suitable materials include medical grade plastics, suchas polycarbonates or polymethyl methacrylates, or other acrylates. It isgenerally desirable for the material to be somewhat transparent, so thata clear window can be produced and the internal flow channels and flowpaths can be seen during operation of the acoustophoresis device/system.

Various coatings may be used on the internal flow channels of themodules. Such coatings include epoxies, for example epichlorohydrinbisphenol crosslinked with an amine or a polyamide; or polyurethanecoatings, for example a polyester polyol crosslinked with aliphaticisocyanates. Such coatings are useful for producing a smooth surfaceand/or reducing surface tension, permitting cells to slide better underthe influence of gravity along the flow channel surface and into desiredlocations (such as collection well modules).

The flow rate of the acoustophoretic device must be controlled so thatgravity can act on particle aggregates. In this regard, it iscontemplated that the particle/fluid mixture passing in/out of the flowpath in the acoustophoretic device through the inlet/outlet modules orthe port module can flow at rates of up to about 100 milliliters perminute (ml/min). By way of comparison, the flow rate out of thecollection well modules through the ports is much less, from about 3ml/min up to about 10 ml/min.

The present disclosure contemplates kits formed from any combination ofthe modules described above. In particular embodiments, the kits includeat least an ultrasonic transducer module 200, a collection well module300/500, and an inlet/outlet module 400/600. In other embodiments, thekits include at least an ultrasonic transducer module 200, a collectionwell module 300/500, two port modules 700, and a three-way connectormodule 892. In yet additional embodiments, the kits include at least twoultrasonic transducer modules 200, at least two collection well modules300/500, a three-way connector module 892, and either (i) aninlet/outlet module 400/600 or two port modules 700.

Various acoustophoretic systems can be made using the different modularcomponents described above. FIGS. 17-29 illustrate different systems. Itis noted that the three-way connector modules used herein have two maleattachment members and one female attachment member, with the sideattachment member being a male member.

The system 1000 of FIG. 17 is built from two port modules 1002, 1004, atwo-way curved connector module 1006, two three-way connector modules1008, 1010, two ultrasonic transducer modules 1012, 1014, two collectionwell modules 1016, 1018, and a female/female two-way connector module1020. Starting at the left, a port module 1002 acts as the inlet, and isconnected to the female/female two-way connector module 1020, which isin turn connected to the two-way curved connector module 1006, whichjoins a three-way connector module 1008. The first collection wellmodule 1016 connects to the three-way connector module 1008, and acts asa base for the system. An ultrasonic transducer module 1012 is thenconnected, then the other three-way connector module 1010. The secondcollection well module 1018 connects to this three-way connector module1010, and also acts as a base. Note the second collection well module1018 is taller than the first collection well module 1016, so that theoverall system is tilted at an acute angle. The second ultrasonictransducer module 1014 is connected, and then the second port module1004 acts as an outlet. Fluid flow is in the direction of the arrow1005. In operation, as particle aggregates grow in each ultrasonictransducer module 1012, 1014 and fall out of the acoustic standing wavefield, they follow gravity downwards and countercurrent to the fluidflow into their respective collection well module 1016, 1018. Anyparticles that escape the first ultrasonic transducer module 1012 shouldbe trapped by the second ultrasonic transducer module 1014. It iscontemplated that as a result, the size of the particle aggregates inthe first ultrasonic transducer module should generally be differentfrom the aggregates in the second ultrasonic transducer module, as theaggregates in the second ultrasonic transducer module should grow at aslower rate due to the lower number of particles in the fluid passingthrough.

FIG. 18 is a relatively simple system 1000 built from two port modules1002, 1004, a three-way connector module 1008, an ultrasonic transducermodule 1012, a collection well module 1016, and a female/female two-wayconnector module 1020. The particle/fluid mixture flows into the systemthrough port module 1002, which acts as an inlet, and the female/femaletwo-way connector module 1020. Particle aggregates can be swept by thecurrent into the three-way connector module 1008, where they then fallinto the collection well module 1016. Fluid flows up and out of thesystem through port module 1004, which acts as an outlet.

FIG. 19 is a variation on the system of FIG. 18. Here, a secondultrasonic transducer module 1014 is added before the port module 1004.Aggregates formed in the second ultrasonic transducer module 1014 canfall directly into the collection well device 1016.

The system of FIG. 20 uses 11 different modules. Here, theparticle/fluid mixture enters the system through port module 1002.Particles are trapped by the first ultrasonic transducer module 1012.Any particles that escape the first ultrasonic transducer module shouldbe trapped by the second ultrasonic transducer module 1014. In contrastto the system of FIG. 17, though, the aggregates from the firstultrasonic transducer module will also pass through the secondultrasonic transducer module. This should sweep all aggregates out andinto the collection well module. Fluid can exit the system at the topthrough port module 1004. A third port module 1022 is provided betweenthe ultrasonic transducers 1012, 1014 and the collection well module1016. This third port module 1022 also acts as an outlet, and can beused for draining fluid if desired, or for handling overflow from thecollection well module (e.g. if particles build up too quickly to becompletely drained through the port).

FIG. 21 is similar to FIG. 20, except that there is no curved two-wayconnector module 1006 between the two ultrasonic transducer modules1012, 1014. Only 10 modules are used.

FIG. 22 is a simple system that uses an ultrasonic transducer module1012, a collection well module 1016, and an inlet/outlet module 1024.The particle/fluid mixture flows through one sub-channel 1026 into theultrasonic transducer module 1012, and pressure pushes fluid out theother sub-channel 1028. Particle aggregates fall directly into thecollection well module 1016.

In FIG. 23, the particle/fluid mixture enters through port module 1002and fluid exits the system through port module 1004. Here, the particleaggregates from both ultrasonic transducers 1012, 1014 is collected inthe same (and only) collection well module 1016.

In FIG. 24, the particle/fluid mixture enters through port module 1002and fluid exits the system through port module 1004. Fluid flow is in aU-shape. The particle aggregates from each ultrasonic transducer 1012,1014 fall directly downwards into a collection well module 1016, 1018.Also, a male/male two-way connector module 1036 is used between modules1010, 1018.

The system of FIG. 25 is similar to the system of FIG. 24, but adds athird ultrasonic transducer 1030. A male/male two-way connector module1036 is used between the modules 1010, 1018.

The system of FIG. 26 illustrates the use of the angled collection wellmodule 1032. The particle/fluid mixture enters through port module 1002and fluid exits the system through port module 1004. The particleaggregates from both ultrasonic transducer modules 1012, 1014 settle bygravity into the angled collection well module 1032 and move down to theport 1033.

FIG. 27 illustrates a system with another collection well module 1016and a third ultrasonic transducer module 1030 added to the system ofFIG. 26, but otherwise operates in the same manner. The particleaggregates from the ultrasonic transducer modules 1012, 1014 stillsettle by gravity into the angled collection well module 1032.

In FIG. 28, a curved two-way connector module 1006 turns the fluid flowpath of FIG. 27 into a horizontal orientation. Here, the particleaggregates from ultrasonic transducer module 1012 still settle bygravity into the angled collection well module. The particle aggregatesfrom ultrasonic transducer module 1018 are now captured in a thirdcollection well module 1034.

In FIG. 29, the particle/fluid mixture flows into a three-way connector1008 and then travels upwards into ultrasonic transducer module 1012 andout through port module 1002. Particle aggregates fall downwards into avariable-volume collection module 1016.

This collection module is formed from a housing having an upper end andan opposing lower end. An inlet is located at the upper end of thehousing, and leads to a well. The well has a constant cross-section. Aplunger provides a floor to the well, with the plunger adapted to movethrough the well from the bottom end towards the upper end. Here, thecollection module is in the form of a large cylinder. A plunger 1035 ispresent at the bottom of the collection module. This can be used tocompact the particle aggregates by moving the plunger upwards, reducingthe volume of the well. The particle aggregates remain against theplunger. Any particles still suspended in the fluid will either join theaggregates against the plunger, or be pushed into the acoustic standingwave field in the ultrasonic transducer module, and so are not lostthrough the outlet 1002. The particles can then be collected. It iscontemplated that this module can be used for batch processing.Alternatively, a port (not illustrated) can located on a side of thehousing proximate the upper end, which is fluidly connected to the well.The particles can be collected through the port.

Some explanation of the ultrasonic transducers used in the devices ofthe present disclosure may be useful as well. In this regard, thetransducers use a piezoelectric crystal, usually made of PZT-8 (leadzirconate titanate). Such crystals may have a 1 inch diameter and anominal 2 MHz resonance frequency. Each ultrasonic transducer module canhave only one crystal, or can have multiple crystals that each act as aseparate ultrasonic transducer and are either controlled by one ormultiple amplifiers.

FIG. 30 is a cross-sectional diagram of a conventional ultrasonictransducer. This transducer has a wear plate 50 at a bottom end, epoxylayer 52, ceramic crystal 54 (made of, e.g. PZT), an epoxy layer 56, anda backing layer 58. On either side of the ceramic crystal, there is anelectrode: a positive electrode 61 and a negative electrode 63. Theepoxy layer 56 attaches backing layer 58 to the crystal 54. The entireassembly is contained in a housing 60 which may be made out of, forexample, aluminum. An electrical adapter 62 provides connection forwires to pass through the housing and connect to leads (not shown) whichattach to the crystal 54. Typically, backing layers are designed to adddamping and to create a broadband transducer with uniform displacementacross a wide range of frequency and are designed to suppress excitationat particular vibrational eigen-modes. Wear plates are usually designedas impedance transformers to better match the characteristic impedanceof the medium into which the transducer radiates.

FIG. 31 is a cross-sectional view of an ultrasonic transducer 81 of thepresent disclosure. Transducer 81 has an aluminum housing 82. A PZTcrystal 86 defines the bottom end of the transducer, and is exposed fromthe exterior of the housing. The crystal is supported on its perimeterby a small elastic layer 98, e.g. silicone or similar material, locatedbetween the crystal and the housing. Put another way, no wear layer ispresent.

Screws (not shown) attach an aluminum top plate 82 a of the housing tothe body 82 b of the housing via threads 88. The top plate includes aconnector 84 to pass power to the PZT crystal 86. The bottom and topsurfaces of the PZT crystal 86 are each connected to an electrode(positive and negative), such as silver or nickel. A wrap-aroundelectrode tab 90 connects to the bottom electrode and is isolated fromthe top electrode. Electrical power is provided to the PZT crystal 86through the electrodes on the crystal, with the wrap-around tab 90 beingthe ground connection point. Note that the crystal 86 has no backinglayer or epoxy layer as is present in FIG. 30. Put another way, there isan air gap 87 in the transducer between aluminum top plate 82a and thecrystal 86 (i.e. the air gap is completely empty). A minimal backing 58and/or wear plate 50 may be provided in some embodiments, as seen inFIG. 32.

The transducer design can affect performance of the system. A typicaltransducer is a layered structure with the ceramic crystal bonded to abacking layer and a wear plate. Because the transducer is loaded withthe high mechanical impedance presented by the standing wave, thetraditional design guidelines for wear plates, e.g., half wavelengththickness for standing wave applications or quarter wavelength thicknessfor radiation applications, and manufacturing methods may not beappropriate. Rather, in one embodiment of the present disclosure thetransducers, there is no wear plate or backing, allowing the crystal tovibrate in one of its eigenmodes with a high Q-factor. The vibratingceramic crystal/disk is directly exposed to the fluid flowing throughthe flow chamber.

Removing the backing (e.g. making the crystal air backed) also permitsthe ceramic crystal to vibrate at higher order modes of vibration withlittle damping (e.g. higher order modal displacement). In a transducerhaving a crystal with a backing, the crystal vibrates with a moreuniform displacement, like a piston. Removing the backing allows thecrystal to vibrate in a non-uniform displacement mode. The higher orderthe mode shape of the crystal, the more nodal lines the crystal has. Thehigher order modal displacement of the crystal creates more trappinglines, although the correlation of trapping line to node is notnecessarily one to one, and driving the crystal at a higher frequencywill not necessarily produce more trapping lines.

In some embodiments, the crystal may have a backing that minimallyaffects the Q-factor of the crystal (e.g. less than 5%). The backing maybe made of a substantially acoustically transparent material such asbalsa wood, foam, or cork which allows the crystal to vibrate in ahigher order mode shape and maintains a high Q-factor while stillproviding some mechanical support for the crystal. The backing layer maybe a solid, or may be a lattice having holes through the layer, suchthat the lattice follows the nodes of the vibrating crystal in aparticular higher order vibration mode, providing support at nodelocations while allowing the rest of the crystal to vibrate freely. Thegoal of the lattice work or acoustically transparent material is toprovide support without lowering the Q-factor of the crystal orinterfering with the excitation of a particular mode shape.

Placing the crystal in direct contact with the fluid also contributes tothe high Q-factor by avoiding the dampening and energy absorptioneffects of the epoxy layer and the wear plate. Other embodiments mayhave wear plates or a wear surface to prevent the PZT, which containslead, contacting the host fluid. This may be desirable in, for example,biological applications such as separating blood. Such applicationsmight use a wear layer such as chrome, electrolytic nickel, orelectroless nickel. Chemical vapor deposition could also be used toapply a layer of poly(p-xylylene) (e.g. Parylene) or other polymer.Organic and biocompatible coatings such as silicone or polyurethane arealso usable as a wear surface.

In the present systems, the system is operated at a voltage such thatthe particles are trapped in the ultrasonic standing wave, i.e., remainin a stationary position. The particles are collected in along welldefined trapping lines, separated by half a wavelength. Within eachnodal plane, the particles are trapped in the minima of the acousticradiation potential. The axial component of the acoustic radiation forcedrives the particles, with a positive contrast factor, to the pressurenodal planes, whereas particles with a negative contrast factor aredriven to the pressure anti-nodal planes. The radial or lateralcomponent of the acoustic radiation force is the force that traps theparticle. The radial or lateral component of the acoustic radiationforce is on the same order of magnitude as the axial component of theacoustic radiation force. As discussed above, the lateral force can beincreased by driving the transducer in higher order mode shapes, asopposed to a form of vibration where the crystal effectively moves as apiston having a uniform displacement. The acoustic pressure isproportional to the driving voltage of the transducer. The electricalpower is proportional to the square of the voltage.

In embodiments, the pulsed voltage signal driving the transducer canhave a sinusoidal, square, sawtooth, or triangle waveform; and have afrequency of 500 kHz to 10 MHz. The pulsed voltage signal can be drivenwith pulse width modulation, which produces any desired waveform. Thepulsed voltage signal can also have amplitude or frequency modulationstart/stop capability to eliminate streaming.

The size, shape, and thickness of the transducer determine thetransducer displacement at different frequencies of excitation, which inturn affects separation efficiency. Typically, the transducer isoperated at frequencies near the thickness resonance frequency (halfwavelength). Gradients in transducer displacement typically result inmore places for particles to be trapped. Higher order modaldisplacements generate three-dimensional acoustic standing waves withstrong gradients in the acoustic field in all directions, therebycreating equally strong acoustic radiation forces in all directions,leading to multiple trapping lines, where the number of trapping linescorrelate with the particular mode shape of the transducer.

FIG. 33 shows the measured electrical impedance amplitude of thetransducer as a function of frequency in the vicinity of the 2.2 MHztransducer resonance when operated in a water column containing oildroplets. The minima in the transducer electrical impedance correspondto acoustic resonances of the water column and represent potentialfrequencies for operation. Numerical modeling has indicated that thetransducer displacement profile varies significantly at these acousticresonance frequencies, and thereby directly affects the acousticstanding wave and resulting trapping force. Since the transduceroperates near its thickness resonance, the displacements of theelectrode surfaces are essentially out of phase. The typicaldisplacement of the transducer electrodes is not uniform and variesdepending on frequency of excitation. As an example, at one frequency ofexcitation with a single line of trapped oil droplets, the displacementhas a single maximum in the middle of the electrode and minima near thetransducer edges. At another excitation frequency, the transducerprofile has multiple maxima leading to multiple trapped lines of oildroplets. Higher order transducer displacement patterns result in highertrapping forces and multiple stable trapping lines for the captured oildroplets.

To investigate the effect of the transducer displacement profile onacoustic trapping force and oil separation efficiencies, an experimentwas repeated ten times, with all conditions identical except for theexcitation frequency. Ten consecutive acoustic resonance frequencies,indicated by circled numbers 1-9 and letter A on FIG. 33, were used asexcitation frequencies. The conditions were experiment duration of 30min, a 1000 ppm oil concentration of approximately 5-micron SAE-30 oildroplets, a flow rate of 500 ml/min, and an applied power of 20W.

As the emulsion passed by the transducer, the trapping lines of oildroplets were observed and characterized. The characterization involvedthe observation and pattern of the number of trapping lines across thefluid channel, as shown in FIG. 34A, for seven of the ten resonancefrequencies identified in FIG. 33.

FIG. 34B shows an isometric view of the ultrasonic transducer volume inwhich the trapping line locations are being determined. FIG. 34C is aview of the ultrasonic transducer volume as it appears when looking downthe inlet, along arrow 114. FIG. 34D is a view of the ultrasonictransducer volume as it appears when looking directly at the transducerface, along arrow 116.

The effect of excitation frequency clearly determines the number oftrapping lines, which vary from a single trapping line at the excitationfrequency of acoustic resonance 5 and 9, to nine trapping lines foracoustic resonance frequency 4. At other excitation frequencies four orfive trapping lines are observed. Different displacement profiles of thetransducer can produce different (more) trapping lines in the standingwaves, with more gradients in displacement profile generally creatinghigher trapping forces and more trapping lines.

Finally, FIG. 35 is a lin-log graph (linear y-axis, logarithmic x-axis)that shows the scaling of the acoustic radiation force, fluid dragforce, and buoyancy force with particle radius. Calculations are donefor a typical SAE-30 oil droplet used in experiments. The buoyancy forceis a particle volume dependent force, and is therefore negligible forparticle sizes on the order of micron, but grows, and becomessignificant for particle sizes on the order of hundreds of microns. Thefluid drag force scales linearly with fluid velocity, and thereforetypically exceeds the buoyancy force for micron sized particles, but isnegligible for larger sized particles on the order of hundreds ofmicrons. The acoustic radiation force scaling acts differently. When theparticle size is small, the acoustic trapping force scales with thevolume of the particle. Eventually, when the particle size grows, theacoustic radiation force no longer increases with the cube of theparticle radius, and will rapidly vanish at a certain critical particlesize. For further increases of particle size, the radiation forceincreases again in magnitude but with opposite phase (not shown in thegraph). This pattern repeats for increasing particle sizes.

Initially, when a suspension is flowing through the system withprimarily small micron sized particles, it is necessary for the acousticradiation force to balance the combined effect of fluid drag force andbuoyancy force for a particle to be trapped in the standing wave. InFIG. 35 this happens for a particle size of about 3.5 micron, labeled asR_(c1). The graph then indicates that all larger particles will betrapped as well. Therefore, when small particles are trapped in thestanding wave, particles coalescence/clumping/aggregation/agglomerationtakes place, resulting in continuous growth of effective particle size.As the particle size grows, the acoustic radiation force reflects offthe particle, such that large particles will cause the acousticradiation force to decrease. Particle size growth continues until thebuoyancy force becomes dominant, which is indicated by a second criticalparticle size, R_(c2), at which size the particles will rise or sink,depending on their relative density with respect to the host fluid. Asthe particles rise or sink, they no longer reflect the acousticradiation force, so that the acoustic radiation force then increases.Not all particles will drop out, and those remaining particles willcontinue to grow in size as well. This phenomenon explains the quickdrops and rises in the acoustic radiation force beyond size R_(c2).Thus, FIG. 35 explains how small particles can be trapped continuouslyin a standing wave, grow into larger particles or clumps, and theneventually will rise or settle out because of increased buoyancy force.

The acoustophoretic devices of the present disclosure can beincorporated into acoustophoretic systems in which the devices arearranged in series to create a filter “train.” The systems can thereforebe considered multi-stage systems. The use of multiple stages ofacoustic filtration reduces the burden on subsequent filtration stagesand provides for (i) better clarification of the fluid and (ii) betterseparation of cells from the fluid. This permits recovery of the cellsand/or the clarified fluid, as desired.

One embodiment of a multi-stage acoustophoretic system 3600 is shown inFIG. 36. The system 3600 is a four-stage system incorporating fouracoustophoretic devices 3610. Each device 3610 can be considered adifferent clarification stage. Each acoustophoretic device 3610 can beconstructed as described herein with a transducer-reflector pair(s) tocreate at least one multi-dimensional acoustic standing wave within eachdevice. The acoustic chambers of each device can, in certainembodiments, be 1″×2″. The nominal flow rate through the system can beabout 4 L/hour.

In the system 3600 depicted in FIG. 36, the stages 3610 are connected toeach other in series, with each device/stage 3610 being connected toadjacent stages by tubing 3620 running therebetween. Attaching adjacentstages to one another using tubing (as opposed to directly connectingthe device of each stage to adjacent devices) provides for betterseparation of fluid and particular. A pump 3630 may be provided betweeneach device, and additional pumps can be provided upstream of the firstdevice and downstream of the last device. In this regard, it is notedthat in the four-stage system 3600 of FIG. 36, five pumps 3630 would beused for the four devices/stages 3610. In addition, a flowmeter 3640 ispresent adjacent each pump 3630. As illustrated here, five flowmetersare present. Thus, fluid flow is through a feed pump, then a flowmeter,then the first clarification stage, then a pump, then a flowmeter, etc.,ending with a final pump and a flowmeter.

As illustrated in FIG. 37A, the individual device stages 3610 arephysically located side-by-side, and are physically connected to eachother. This is not required—the stages can be separated from each other,and simply be fluidly connected by tubing. FIG. 37B is a rear view of asingle stage 3610.

The multi-dimensional acoustic standing wave(s) created in eachacoustophoretic device of the multi-stage acoustophoretic system canhave a different frequency from the other stage(s), either higher orlower, which can be particularly useful for separating particles ofdifferent sizes or contrast factors or for otherwise selectivelyfiltering certain particles. The frequencies of the multiple acousticstanding waves can be varied as desired to send different sizes and/orconcentrations of materials to different stages of the system, therebyimproving the efficiency of the process. The frequency of any stage canbe equal to, or different from, each other stage as well. It isparticularly contemplated that the frequencies of all of the stages arewithin one order of magnitude of each other.

FIG. 37C shows a cross-sectional diagram of one of the device/stages3610. This device can be used to ameliorate some of the problems with afluid at low particle Reynolds numbers, and create a more uniform flowthrough the device. The device 3610 has upward, vertical flow throughthe acoustic chamber. The acoustic chamber also has two opposing dumpdiffusers 3612 and a collector design which provides a vertical plane orline of flow symmetry. Generally, the cross-section of the device in theflow direction is circular or rectangular. The acoustic chamber isempty, i.e. there is nothing within the chamber, and fluid flows throughthe acoustic chamber. At least one permeate outlet 3614 is present atthe upper end of the acoustic chamber. At least one solids outlet 3616is present at the lower end of the acoustic chamber. A shallow wall 3618is present at the lower end of the acoustic chamber, and leads to thesolids outlet 3616. The shallow wall is angled relative to a horizontalplane, such as the bottom of the acoustic chamber. At least oneultrasonic transducer (not shown) is present on a sidewall of theacoustic chamber, and at least one reflector (not shown) is present onthe sidewall opposite the ultrasonic transducer.

This device 3610 includes a symmetrical, dual dump diffuser, plenuminlet configuration. Here, two dump diffusers 3612 are placed onopposite sides of the device. Each dump diffuser has a plenum/chamberwith an upper end 3620 and a lower end 3622. The plenum volume providesflow diffusion and dramatically reduces incoming flow non-uniformities.An inlet flow port 3624 is located above the lower end 3622, and atleast one flow outlet 3626 is located at the lower end of the plenum. Asolid wall 3628 is present at the upper end of the plenum. These dumpdiffuser flow outlets can be in the form of slots or a line of holes,and they are placed above the bottom of the acoustic chamber. Thediffusers 3612 provide a flow direction normal to the axial direction ofthe acoustic standing waves generated by the ultrasonic transducer. Theacoustic chamber inlets are also arranged so that they are in opposinglocations, so that the horizontal velocity will decrease to zero in thecenter of the acoustic chamber.

The dump diffusers eliminate downward flow in the acoustic chamber. Themixture fills up the plenum in the dump diffuser and then flowshorizontally into the acoustic chamber, where the mixture flowsvertically upwards past the acoustic standing waves. The dump diffuserreduces/eliminates flow pulsations and flow non-uniformities that resultfrom pumps, hosing and horizontal inlet flow where gravity effectsdominate. The dump diffuser brings the heavier mixture into the acousticchamber below the ultrasonic transducer and the nodal clusters that formin the ultrasonic standing waves. This minimizes any disturbances of theclusters set up by inflowing material.

The vertical plane or line of symmetry is aligned with gravity forces.Also shown are flow streamlines which are desirably symmetrical, sincethis minimizes non-uniformities, eddy disturbances, circulation, anddisturbance of clusters falling through the solids outlet 3616 to becollected. Symmetry also maximizes gravity forces in the inlet flowdistribution and particle collection process. Because it is heavier thanthe permeate exiting at the top of the device, the (relatively) heavyincoming mixture comes in near the bottom of the acoustic chamber,spreads out across the bottom of the chamber due to gravity forces, andprovides near uniform velocity profiles from bottom to top. Thehorizontal velocity of the mixture will decrease to zero as itapproaches the center of the acoustic chamber due to the dual opposinginlet flows. This assures minimum interference between the chamber flowand dropping particle clusters. A uniform velocity provides the bestseparation and collection results because the lateral acoustic forceshave to overcome particle drag for the clusters to grow and continuouslydrop out of the acoustic chamber. This also eliminates the need for aninlet flow distributor.

As the particle clusters drop out, the axial acoustic forces associatedwith the standing wave will keep the clusters intact. This assures rapiddropping of the clusters with high terminal velocities, on the order of1 cm/sec. This is extremely fast compared to the chamber flowvelocities, which are on the order of 0.1 cm/sec to 0.3 cm/sec. Theshallow wall angle means the cylindrical particle clusters have to droponly a very short distance before they exit the acoustic chamber, sothat little dispersion of the clusters occurs. Ideally, the systemoperates with 3 to 12 crystal vibration nodes per square inch oftransducer. The symmetry, minimum flow disturbance in the centralcollection region, and shallow collector walls provide good collectionwithout the need for baffles/laminar plates.

The present disclosure will further be illustrated in the followingnon-limiting working examples, it being understood that these examplesare intended to be illustrative only and that the disclosure is notintended to be limited to the modules, devices, conditions, processparameters and the like recited herein.

EXAMPLES

Various mixtures of CHO cells in cell culture media were filtered.

Example 1

The acoustophoretic separation process was compared to depth flowfiltration (DFF). First, a baseline of DFF capacity was obtained byperforming two rounds of clarification, a primary clarification and asecondary clarification. The setup for this baseline is illustrated inFIG. 38.

The pressure drop was measured during the two rounds. The separationapparatus was operated at 145 LMH (liters/m²/hour). The pressure wasmeasured at three different locations P1, P2, and P3. Located betweeneach set of sensors was a filter. The filter used during the primaryclarification was a D0HC filter, and the filter used in the secondaryclarification was a X0HC filter, both available from Millipore.

A mixture of CHO cells and culture media were flowed through thefilters, and the permeate was then collected in a tank. The CHO cellswere removed by the filters. The feed had a total cell density (TCD) of6.34×10⁶ cells/mL and a turbidity of 815 NTU. The final permeate in thethird tank had a turbidity of 1.75 NTU.

FIG. 38 is a performance graph showing the pressure drop versusvolumetric throughput capacity. The pressure drop in the primary filterwas lowest at low throughput, then became greater than the pressure dropin the secondary filter above approximately 88 L/m² capacity. The totalpressure drop is the top line in the graph. A pressure drop of 15 psigwould be attained at a volumetric throughput of 88 L/m² (indicated bydashed lines). This indicates that if scaled up with a maximum pressuredrop of 15 psig (Pmax=15 psig), then the area of the filter for theprimary clarification and for the secondary clarification would eachneed to be 11.4 m².

Example 2

Next, the two-step DFF described in Example 1 was compared to a two-stepclarification process in which the primary clarification was performedby acoustic wave separation (AWS) and the secondary clarification wasperformed by DFF. This is described in FIG. 39.

As indicated there, in the two-step DFF, each filter had an area of 11m². Each filter was operated with a pressure drop of 7.5 psig. Thevolumetric throughput (VT) at 7.5 psig (VT_(7.5)) was 84 L/m² for eachfilter.

The acoustophoretic system used to perform the AWS was made up of threeacoustophoretic devices as illustrated in FIG. 8 and linked in series,such as those depicted in FIG. 36. The transducer in each device was 1inch by 1 inch. The system had a total acoustic volume of 49 cm³. TheAWS system was paired with a DFF filter having a total area of 6 m².Because there is no pressure drop in the AWS system, the DFF filtercould be operated at a pressure drop of 15 psig, resulting in a VT₁₅ of160 L/m².

The feed had a total cell density (TCD) of 6.7×10⁶ cells/mL and aturbidity of 835 NTU, and 77% cell viability. The feed rate to theacoustophoretic system was 4 kg at 2.5 liters per hour (LPH).

The results for the primary clarification using the AWS system are shownin FIG. 39. The acoustophoretic system achieved 91% TCD reduction, 90%turbidity reduction, and 91.2% recovery of protein. The graph at thebottom left is percent reduction versus time, and shows that the AWSsystem operated consistently during the test.

Example 3

The same experiment as described in Example 2 was performed again, butwith a higher cell density. The feed had a higher TCD of 15.6×10⁶cells/mL and a turbidity of 3608 NTU, and 68% cell viability. This isdescribed in FIG. 40.

The two-step DFF process used filters of 38 m² and 17 m², respectively.As indicated, the VT₇₅ was 26 L/m² for the primary clarification and 58L/m² for the secondary clarification. In the AWS-DFF process, the AWSsystem had only two acoustophoretic devices in series (not three as inExample 2), with a total acoustic volume of 33 cm³. The DFF filter had atotal area of 11 m², and a VT₁₅ of 85 L/m². The feed rate to theacoustophoretic system was 8 kg at 2.5 liters per hour (LPH).

The results for the primary clarification using the AWS system are shownin FIG. 38. The acoustophoretic system achieved 94% TCD reduction, 91%turbidity reduction, and 92% recovery of protein. The graph at thebottom left is percent reduction versus time, and shows that the systemoperated consistently during the test. Higher cell densities were moredifficult for the DFF device, as indicated by the lower VT. However, theacoustophoretic device was able to handle the higher density with a muchlower reduction in VT.

Example 4

The feed had a TCD of 7.5×10⁶ cells/mL and a turbidity of 819 NTU, and88% cell viability. Clarification was performed using a three-stageacoustophoretic system as in Example 1.

The first stage reduced the cell density by 62%. The second stagereduced the remaining cell density by 87% (cumulative 95%). The thirdstage reduced the remaining cell density by 63% (cumulative 98%). Onlytwo stages were needed to attain greater than 90% cell densityreduction.

The first stage reduced the turbidity by 68% from 819 NTU to 260 NTU.The second stage reduced the remaining turbidity down to 54 NTU(cumulative 94%). The third stage reduced the remaining turbidity to 42NTU (cumulative 95%). Only two stages were needed to attain greater than90% turbidity reduction. This is important for secondary filtrationprocesses further downstream.

The percent reduction for both cell density reduction and turbidityreduction was consistent over the entire time, meaning the deviceoperated well on a continuous basis. Again, these are both important forsecondary filtration processes further downstream, and for ultimatelythe chromatographic separation of monoclonal antibodies or recombinantproteins from the clarified fluid.

Example 5

Five different lots were tested through the three-stage system ofExample 1. Each lot had its own cell size and density characteristics.The feeds had a TCD of 7 to 8.5×10⁶ cells/mL, a turbidity of 780 to 900NTU, and 82% to 93% cell viability. This example tested the consistencyof performance of the system across different batches.

Over the five different lots, the turbidity of the permeate was reduced84% to 86%, with a standard deviation of 1% after three passes. The celldensity of the permeate was reduced 93% to 97%, with a standarddeviation of 2% after three passes.

In other experiments not described here, it was found that the acousticwave separation processes using a multi-dimensional acoustic standingwave did not affect the physical or chemical characteristics of proteinor monoclonal antibodies recovered from the permeate.

Example 6

Next, the effect of voltage input on clarification performance wasobserved in a three-stage or a four-stage acoustophoretic device such asin FIG. 36.

A mixture of CHO cells and culture media were flowed through the stagesof the device, and the permeate was then collected in a tank. The CHOcells were removed by the filters. The feed had a total cell density(TCD) of 25×10⁶ cells/mL, a turbidity of 2048 NTU, and a cell viabilityof 72%. The feed flow rate was 30 mL/min and the solids draws were 2.34mL/min for stage one, 1.41 mL/min for stage two, 0.94 mL/min for stagethree, and 0 mL/min for stage four.

The TCD reduction after three and four stages of filtering are shown inFIG. 41. In this Figure T1 refers to the voltage in the first stage, T2to the voltage in the second stage, T3 to the voltage in the thirdstage, and T4 to the voltage in the fourth stage. Five different testruns using different voltages in different stages are shown. For allvoltage conditions of 50V and 60V, the system achieved a greater than90% cell density reduction. The addition of the fourth stage yielded ahigh 95% reduction, lowering the TCD from 2.9×10⁶ cells/mL after threestages to 1.3×10⁶ cells/mL after four stages. As can be seen in FIG. 42,voltages of 50V and 60V yielded the best performances through eachstage. As can be seen in FIG. 43, adding a fourth stage at 50V increasedthe TCD reduction by 10% aggregate. Generally, a voltage of 50V to 60Vshould be used in the first and/or second stages to obtain high TCDreduction.

The total turbidity reduction after three and four stages of filteringare shown in FIG. 44. For all voltage conditions of 50V and 60V, thesystem achieved a greater than 90% turbidity reduction. The addition ofthe fourth state yielded a 94% reduction, lowering permeate from 390 NTUafter three stages to 177 NTU after four stages.

In summary, the 50V and 60V operating conditions yielded higher andnear-equivalent clarification over the 40V operating condition, and useof the 40V operating condition in the third stage after 50V/60V in thefirst and second stages lowered the clarification efficiency.

Example 7

Next, similar to Example 1, the acoustophoretic separation process wascompared to depth flow filtration (DFF) to determine the effect of DFFperformance on acoustic wave separation (AWS) performance.

A mixture of CHO cells and culture media were flowed through twofilters, a D1HC filter and a X0HC filter, both available from Millipore,and the permeate was then collected in a tank. The CHO cells wereremoved by the filters. The feed had a total cell density (TCD) of24.7×10⁶ cells/mL and a turbidity of 2850 NTU. The final permeate in thetank had a turbidity of 4.9 NTU.

FIG. 45 is a performance graph showing the pressure drop versusvolumetric throughput capacity. The profiles in FIG. 45 were plotted perstage based on the total filter area in each stage. The volumetricthroughput capacity was calculated at 23 cm². The pressure drop in theD0HC filter was lowest at low throughput, then became greater than thepressure drop in the X0HC filter above approximately 16 L/m² capacity. Apressure drop of 15 psig would be attained at a volumetric throughput of47 L/m² for the D0HC filter and approximately 110 L/m² for the D0HCfilter (estimated based on pressure-drop ratios—the D0HC filteraccounting for 12.0 psig of the total 15.0 psig pressure drop and theX0HC filter accounting for the remaining 3.0 psig).

FIG. 46 is a performance graph showing the pressure drop versusvolumetric throughput capacity. The profiles in FIG. 46 were plotted forthe total filter area in series (i.e., across all stages). Thevolumetric throughput capacity was calculated at 46 cm². The pressuredrop in the D0HC filter was lowest at low throughput, then becamegreater than the pressure drop in the X0HC filter above approximately 8L/m² capacity. The total pressure drop is the top line in the graph. Apressure drop of 15 psig would be attained at a volumetric throughput of21.5 L/m². This indicates that if scaled up with a maximum pressure dropof 15 psig (Pmax=15 psig), then the area of the filter for the primaryclarification and secondary clarifications would need to be 46.5 m²total with a D0HC:XOHC filter size ratio of 3:1 (i.e., the D0HC filterwould have an area of 34.9 m² and the X0HC filter would have an area of11.6 m².

Example 8

Next, the two-step DFF described in Examples 6 and 7 were compared to atwo-step clarification process in which the primary clarification wasperformed by acoustic wave separation (AWS) and the secondaryclarification was performed by DFF. This is described in FIG. 47.

As indicated there, in the two-step DFF, the D0HC filter had an area of34.9 m² and the X0HC filter had an area of 11.6 m², for a total area of46.5 m². Each filter was operated with a pressure drop of 15 psig. Thevolumetric throughput (VT) at 15 psig (VT₁₅) was 47 L/m² for the D0HCfilter and 110 L/m² for the X0HC filter. The turbidity of the permeatewas 4.9 NTU.

Two different acoustophoretic systems were used to perform the AWS. Thefirst acoustophoretic system was a three-stage system (i.e., made up ofthree acoustophoretic devices) as illustrated in FIG. 8 and linked inseries, such as is depicted in FIG. 36 or FIG. 37. The secondacoustophoretic system was a four-stage system (i.e., made up of fouracoustophoretic devices) as illustrated in FIG. 36 or FIG. 37 and linkedin series. The transducers used in each device of each system were 1inch by 1 inch.

The first AWS system was paired with a DFF filter having a total area of10.2 m². The area by stage was 7.6 m² (3×) and 2.6 m² (1×). Becausethere is no pressure drop in the AWS system, the DFF filter could beoperated at a pressure drop of 15 psig, resulting in a VT₁₅ of 214 L/m².The feed had a total cell density (TCD) of 2.9×10⁶ cells/mL and aturbidity of 380 NTU. The turbidity of the permeate was 8.8 NTU.

The second AWS system was paired with a DFF filter having a total areaof 4.5 m². The area by stage was 3.4 m² (3×) and 1.1 m² (1×). Again,because there is no pressure drop in the AWS system, the DFF filtercould be operated at a pressure drop of 15 psig, resulting in a VT₁₅ of490 L/m². The feed had a total cell density (TCD) of 1.3×10⁶ cells/mLand a turbidity of 176 NTU. The turbidity of the permeate was 11.4 NTU.

The first AWS system (the three-stage system) reduced the overall filterarea by 78%, with a decrease of 12% in secondary clarification ascompared to DFF for primary clarification (centrifuge is the currentunit operation).

The second AWS system (the four-stage system) reduced the overall filterarea by 90%, with a decrease of 60% in secondary clarification ascompared to DFF-DFF. The acoustophoretic system achieved 91% TCDreduction, 90% turbidity reduction, and 91.2% recovery of protein. Thegraph at the bottom left is percent reduction versus time, and showsthat the AWS system operated consistently during the test.

FIG. 48 shows a summary of the DFF filter performance. Harvest to DFF toDFF (primary and secondary) produced a very low volumetric throughput,so low that to process 1000 L would require almost 50 m² of filter area.The volumetric throughput of the DFF step after the third stage wasincreased by 100% with the addition of a fourth stage. TCD was decreasedfrom 2.9×10⁶ cells/mL to 1.4×10⁶ cells/mL (−50% reduction). Turbiditywas decreased from 338 NTU to 220 NTU (42% reduction). Centrate from thecentrifuge was measured to have a TCD of 0.07×10⁶, a turbidity of 450NTU, and a viable cell density (VCD) of 6%. High shear forces causedcell disruption, thereby increasing turbidity and lowering the VCD ofremaining cells. Single-stage DFF results after four-stage AWS performed50-90% below dual stage (COHC+X0HC).

Example 9

Next, the effect of feed flow rates (Q_(F)), total flow ratios(Q_(s)/Q_(F)), and solids flow ratios (Q_(s#)/Q_(s#)) on clarificationperformance was observed in a multi-stage acoustophoretic system. Thisis described in FIG. 49. As seen here, the Q_(F#) indicates the feedflow rate into a given acoustophoretic stage, the Q_(p#) indicates thepermeate flow rate from the given acoustophoretic stage, and the Q_(s#)indicates the solids flow rate from the given acoustophoretic stage. Thepermeate flow rate of an upstream device is the feed flow rate into thesubsequent acoustophoretic stage (e.g., Q_(P1)=QF₂).

Using ten different cell lines, 135 individual runs were performed usinga mixture of CHO cells and culture media having 27-98% cell viabilities,2.8×10⁶ to 56×10⁶ cells/mL cell densities, 1.4 to 16.5% packed cell mass(PCM), and 30 to 9000 NTU feed turbidity.

FIG. 50 illustrates an exemplary four-stage setup. FIG. 51 shows theturbidity reduction as a function of the feed PCM. FIG. 52 shows theturbidity reduction as a function of the feed flow rate for a total flowratio of 20%. FIG. 53 shows the turbidity reduction as a function of thesolids flow ratio for a total flow ratio of 20%. FIG. 54 shows the PCMas a function of the solids flow ratio. FIG. 55 shows the turbidityreduction as a function of the solids flow ratio. FIG. 56 shows thesolids PCM as a function of the solids flow ratio. The feed flow ratewas found to greatly impact clarification. The total flow ratio wasfound to primarily impact yield, while impacting clarification somewhat.The optimized ratio was found to be (feed PCM/50% PCM solids stream).Decreasing the total flow ratio was found to improve yield and increasesolids packing. The solids flow ratio was found to not have an impact onoverall clarification, but was found to allow control over solidsdistribution between stages. The feed PCM was found to impactclarification and stage efficiency. The optimum feed PCM was found to be5-6%, single stage, and higher % PCM is not an issue with multi-stagesystems.

Example 10

Next, the effect of feed flow rates (Q_(F)) on clarification performancewas observed in three- and four-stage acoustophoretic systems.

A mixture of CHO cells and culture media were flowed through bothsystems as a AWS “train” that was then processed using a DFF filter. TheCHO cells were removed by the filters. The feed had a total cell density(TCD) of 35.8×10⁶ cells/mL, a turbidity of 4457 NTU, a PCM of 9.1%, anda viability of 91.9%.

The three-stage system used two runs with feed flow rates (Q_(F)) of1.0× and 0.66×. The four-stage system used a single run with a feed flowrate (Q_(F)) of 1.0×. FIG. 57 shows the % PCM reductions, turbidityreductions, TCD reductions, and yield percentages. In each series, theleftmost bar represents the 1.0× three-stage system, the middlemost barrepresents the 0.6× three-stage system, and the rightmost bar representsthe 1.0× four-stage system The 0.6× three-stage system performedcomparably to the 1.0× four-stage system, though all runs achieved >85%yield and >˜90% turbidity reduction. Each permeate has a separatefiltration chain. The full data recorded is provided below.

AWS-Clarified Permeates Feed Run #1 Run #2 Run #3 Q_(F) 1.0x 0.6x 1.0xStages 3 3 4 TCD 35.8 5.92 2.72 2.52 (×10⁶ cells/mL) Viability (μm)91.9% 91.4% 90.6%    90% Diameter 17.5 18.1 17.7 17.8 PCM 9.1% 2.4% 1.8%2.3% Turbidity 4457 560 235 319

Again, the AWS performed much better than DFF to DFF and achieved a ˜4×increase in volumetric throughput. The overall filtration is summarizedbelow.

Platform Direct Run #1 Run #2 Run #3 Harvest D0-X0 D0HC 256 369 62.5Results (L/m²) (15 ΔP)   (15 ΔP)  (15 ΔP) X0HC 241 352 150.6 (L/m²) (3ΔP)   (5.9 ΔP) (4.1 ΔP) C0-X0 C0HC 203 226 225 Results (L/m²) (15 ΔP)  (at ~4.8 ΔP) (15 ΔP)  X0HC 186 200 209 (L/m²) (1.02 ΔP) (at ~3.1 ΔP) (2ΔP)  

Example 11

Next, 11 individual runs were performed on a four-stage acoustophoreticsystem.

A mixture of CHO cells and culture media were flowed through the fourstages of the system in series in five different CHO cell lines. Thefeed had a total cell density (TCD) of 9.78×10⁶ to 34.3×10⁶ cells/mL, aturbidity of 920 to 4670 NTU, a PCM of 6.5 to 11%, and a viability of31-91%.

The AWS permeate had a TCD of 0.8×10⁶ to 5.2×10⁶ cells/mL(average=2.9×10⁶ cells/mL), a turbidity of 121 to 453 NTU ((average=236NTU), and a PCM of 1.1 to 2.3% (average=1.6%). The reduction performancewas a TCD reduction of 76% to 95% (average=88%), a turbidity reductionof 86% to 97% (average=94%), and a PCM reduction of 72% to 88%(average=79%). The yield was 84% to 89% (average=86%). FIG. 58 shows thereduction performance as a function of the feed PCM, and FIG. 59 showsthe reduction performance as a function of the feed cell viability.

Example 12

Two CHO cell lines with a multi-specific antibody (MM-131) was flowedthrough a three-stage acoustophoretic system with stages having a 1 inchby 1 inch ultrasonic transducer. The feed had a total cell density (TCD)of 20×10⁶ to 24×10⁶ cells/mL. The first feed line had a 80% cellviability and is depicted as the leftmost bar in each series. The secondfeed line had a 65% cell viability and is depicted as the rightmost barin each series. FIG. 60 shows the total cell reduction, turbidityreduction, and protein recovery.

Example 13

In a four-stage system, the system performance was also measured overtime for each stage. FIG. 61 shows the TCD reduction at each stage andat intervals of 2 hours, 4 hours, 6 hours, 9 hours, and 12.5 hours. Theleftmost bar in each series represents the first stage, the second barfrom the left in each series represents the second stage, the second barfrom the right in each series represents the third stage, and therightmost bar in each series represents the fourth stage. Addition ofthe fourth stage showed an increased TCD reduction at each timeinterval. This graph shows that each acoustophoretic stage maintains itsperformance and its separation ability does not decrease over time, as aphysical filter would.

Example 14

For three discrete customers, the reduction in depth filter area wascompared between DFF only and a four-stage AWS system fed into a DFFfilter. FIG. 62 shows that use of a four-stage AWS system reduced thedepth filter area required between 3- and 10-fold.

Example 14

FIG. 63 is a graph showing the cell density reduction for five differentruns through a three-stage system having transducers of size 1 inch by 1inch. The bottom of each bar indicates the total cell density (cells/mL)and the cell viability. The y-axis is the percent reduction of cellsfrom the feed to the permeate, and a higher value is better. Theseresults show good separation even for high total cell density.

The present disclosure has been described with reference to exemplaryembodiments. Obviously, modifications and alterations will occur toothers upon reading and understanding the preceding detaileddescription. It is intended that the present disclosure be construed asincluding all such modifications and alterations insofar as they comewithin the scope of the appended claims or the equivalents thereof.

1. A multi-stage acoustophoretic system, comprising: three or moreacoustophoretic devices fluidly connected to one another in series, eachacoustophoretic device comprising: a flow chamber having at least oneinlet and at least one outlet; at least one ultrasonic transducercoupled to the flow chamber, the transducer including a piezoelectricmaterial driven by a voltage signal to create a multi-dimensionalacoustic standing wave in the flow chamber; and a reflector oppositefrom the at least one ultrasonic transducer.
 2. The multi-stageacoustophoretic system of claim 1, wherein the three or moreacoustophoretic devices are fluidly connected to one another by tubing.3. The multi-stage acoustophoretic system of claim 1, comprising a totalof four acoustophoretic devices.
 4. The multi-stage acoustophoreticsystem of claim 1, further comprising a feed pump and a pump downstreamof each acoustophoretic device.
 5. The multi-stage acoustophoreticsystem of claim 1, further comprising a flowmeter upstream of the firstacoustophoretic device and a flowmeter downstream of eachacoustophoretic device.
 6. The multi-stage acoustophoretic system ofclaim 1, wherein each acoustophoretic device has at least one dumpdiffuser acting as the inlet into the flow chamber.
 7. A method forcontinuously separating a second fluid or a particulate from a hostfluid, the method comprising: flowing a mixture of the host fluid andthe second fluid or particulate through a multi-stage acoustophoreticsystem, the multi-stage acoustophoretic system comprising three or moreacoustophoretic devices fluidly connected to one another, eachacoustophoretic device comprising: a flow chamber having at least oneinlet and at least one outlet; at least one ultrasonic transducerlocated on a wall of the flow chamber, the transducer including apiezoelectric material driven by a voltage signal to create amulti-dimensional acoustic standing wave in the flow chamber; and areflector located on the wall on the opposite side of the flow chamberfrom the at least one ultrasonic transducer; and sending a voltagesignal to drive the at least one ultrasonic transducer of a first one ofthe three or more acoustophoretic devices to create a firstmulti-dimensional acoustic standing wave therein, such that at least aportion of the second fluid or particulate is continuously trapped inthe first standing wave, with the residual host fluid continuing into asecond one of the three or more acoustophoretic devices; sending avoltage signal to drive the at least one ultrasonic transducer of thesecond one of the three or more acoustophoretic devices to create asecond multi-dimensional acoustic standing wave therein, such that atleast a portion of the second fluid or particulate is continuouslytrapped in the second standing wave, with the residual host fluidcontinuing into a third one of the three or more acoustophoreticdevices; and sending a voltage signal to drive the at least oneultrasonic transducer of the third one of the three or moreacoustophoretic devices to create a third multi-dimensional acousticstanding wave therein, such that at least a portion of the second fluidor particulate is continuously trapped in the third standing wave. 8.The method of claim 7, wherein the first, second, and third acousticstanding waves all have different frequencies from each other.
 9. Themethod of claim 7, wherein the first, second, and third acousticstanding waves all have frequencies within one order of magnitude ofeach other.
 10. The method of claim 7, wherein the second fluid orparticulate is Chinese hamster ovary (CHO) cells, NS0 hybridoma cells,baby hamster kidney (BHK) cells, or human cells; T cells, B cells, or NKcells; peripheral blood mononuclear cells (PBMCs); algae; plant cells,bacteria, viruses, or microcarriers.
 11. The method of claim 7, whereinthe three or more acoustophoretic devices are fluidly connected to oneanother by tubing.
 12. The method of claim 7, wherein the multi-stageacoustophoretic system comprises a total of four acoustophoreticdevices.
 13. The method of claim 7, wherein the voltage signal to atleast one of the three or more acoustophoretic devices is at least 50V.14. The method of claim 7, wherein the voltage signal to each of thethree or more acoustophoretic devices is from 50V to about 60V.
 15. Themethod of claim 7, wherein the voltage signal to the furthest downstreamof the three or more acoustophoretic devices is from 40V to about 60V.